Epidermal keratinocytes migrate through the epidermis up to the granular layer where, on terminal differentiation, they progressively lose organelles and convert into anucleate cells or corneocytes. ...Our report explores the role of autophagy in ensuring epidermal function providing the first comprehensive profile of autophagy marker expression in developing epidermis. We show that autophagy is constitutively active in the epidermal granular layer where by electron microscopy we identified double-membrane autophagosomes. We demonstrate that differentiating keratinocytes undergo a selective form of nucleophagy characterized by accumulation of microtubule-associated protein light chain 3/lysosomal-associated membrane protein 2/p62 positive autolysosomes. These perinuclear vesicles displayed positivity for histone interacting protein, heterochromatin protein 1α, and localize in proximity with Lamin A and B1 accumulation, whereas in newborn mice and adult human skin, we report LC3 puncta coincident with misshaped nuclei within the granular layer. This process relies on autophagy integrity as confirmed by lack of nucleophagy in differentiating keratinocytes depleted from WD repeat domain phosphoinositide interacting 1 or Unc-51 like autophagy activating kinase 1. Final validation into a skin disease model showed that impaired autophagy contributes to the pathogenesis of psoriasis. Lack of LC3 expression in psoriatic skin lesions correlates with parakeratosis and deregulated expression or location of most of the autophagic markers. Our findings may have implications and improve treatment options for patients with epidermal barrier defects.
A single-nucleotide polymorphism (SNP) in exon 4 results in expression of either arginine (72R) or proline (72P) at codon 72 of p53. We demonstrate that the in vitro response of cells exposed to ...anticancer agents is strongly influenced by this SNP in wild-type p53. In inducible systems and in cells expressing the endogenous protein, expression of 72P wild-type p53 results in a predominant G1 arrest, with only a minor apoptosis, at drug concentrations causing extensive apoptosis in cells expressing the 72R wild-type variant. The superior apoptosis-inducing activity of the 72R form correlates with more efficient induction of specific apoptosis-associated genes, and is maximal in the presence of serine 46 (S46). In vivo, the outcome of chemo-radiotherapy of squamous carcinomas is more favourable in cancers retaining a wild-type 72R allele, such cases having higher response rates and longer survival than those with wild-type 72P. Together, these results reveal that this SNP is an important determinant of response to anticancer agents in cells expressing wild-type p53. Analysis of complete p53 genotype (mutation and SNP) merits detailed investigation as a simple means for prediction of treatment response and survival in clinical oncology.
iASPP is one of the most evolutionarily conserved inhibitors of p53, whereas ASPP1 and ASPP2 are activators of p53. We show here that, in addition to the DNA-binding domain, the ASPP family members ...also bind to the proline-rich region of p53, which contains the most common p53 polymorphism at codon 72. Furthermore, the ASPP family members, particularly iASPP, bind to and regulate the activity of p53Pro72 more efficiently than that of p53Arg72. Hence, escape from negative regulation by iASPP is a newly identified mechanism by which p53Arg72 activates apoptosis more efficiently than p53Pro72.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
The protein iASPP (encoded by PPP1R13L) is an evolutionarily conserved p53 inhibitor, the expression of which is often upregulated in human cancers. We have recently shown that iASPP is a crucial ...regulator of epidermal homeostasis. Here, we report that iASPP also acts as autophagy inhibitor in keratinocytes. Our data show that depletion of iASPP protects keratinocytes from apoptosis by modulating the expression of Noxa (also known as PMAIP1). In our model, iASPP expression can affect the fission-fusion cycle, mass and shape of mitochondria. iASPP-silenced keratinocytes display disorganization of cytosolic compartments and increased metabolic stress caused by deregulation of mTORC1 signaling. Moreover, increased levels of lipidated LC3 protein confirmed the activation of autophagy in iASPP-depleted cells. We have identified a novel mechanism modulating autophagy in keratinocytes that relies upon iASPP expression specifically reducing the interaction of Atg5-Atg12 with Atg16L1, an interaction that is essential for autophagosome formation or maturation. Using organotypic culture, we further explored the link between autophagy and differentiation, and we showed that impairing autophagy affects epidermal terminal differentiation. Our data provide an alternative mechanism to explain how epithelial integrity is maintained against environmental stressors and might also improve the understanding of the etiology of skin diseases that are characterized by defects in differentiation and DNA damage responses.
Eukaryotic nuclei are essential organelles, storing the majority of the cellular DNA, comprising the site of most DNA and RNA synthesis, controlling gene expression and therefore regulating cellular ...function. The majority of mammalian cells retain their nucleus throughout their lifetime, however, in three mammalian tissues the nucleus is entirely removed and its removal is essential for cell function. Lens fibre cells, erythroblasts and epidermal keratinocytes all lose their nucleus in the terminal differentiation pathways of these cell types. However, relatively little is known about the pathways that lead to complete nuclear removal and about how these pathways are regulated. In this review, we aim to discuss the current understanding of nuclear removal mechanisms in these three cell types and expand upon how recent studies into nuclear degradation in keratinocytes, an easily accessible experimental model, could contribute to a wider understanding of these molecular mechanisms in both health and pathology.
Cutaneous squamous cell carcinomas (cSCCs) account for about 20% of keratinocyte carcinomas, the most common cancer in the UK. Therapeutic options for cSCC patients who develop metastasis are limited ...and a better understanding of the biochemical pathways involved in cSCC development/progression is crucial to identify novel therapeutic targets. Evidence indicates that the phosphoinositide 3-kinases (PI3Ks)/Akt pathway plays an important role, in particular in advanced cSCC. Questions remain of whether all four PI3K isoforms able to activate Akt are involved and whether selective inhibition of specific isoform(s) might represent a more targeted strategy. Here we determined the sensitivity of four patient-derived cSCC cell lines to isoform-specific PI3K inhibitors to start investigating their potential therapeutic value in cSCC. Parallel experiments were performed in immortalized keratinocyte cell lines. We observed that pan PI3Ks inhibition reduced the growth/viability of all tested cell lines, confirming the crucial role of this pathway. Selective inhibition of the PI3K isoform p110α reduced growth/viability of keratinocytes and of two cSCC cell lines while affecting the other two only slightly. Importantly, p110α inhibition reduced Akt phosphorylation in all cSCC cell lines. These data indicate that growth and viability of the investigated cSCC cells display differential sensitivity to isoform-specific PI3K inhibitors.
iASPP, an inhibitory member of the ASPP (apoptosis stimulating protein of p53) family, is an evolutionarily conserved inhibitor of p53 which is frequently upregulated in human cancers. However, ...little is known about the role of iASPP under physiological conditions. Here, we report that iASPP is a critical regulator of epithelial development. We demonstrate a novel autoregulatory feedback loop which controls crucial physiological activities by linking iASPP to p63, via two previously unreported microRNAs, miR‐574‐3p and miR‐720. By investigating its function in stratified epithelia, we show that iASPP participates in the p63‐mediated epithelial integrity program by regulating the expression of genes essential for cell adhesion. Silencing of iASPP in keratinocytes by RNA interference promotes and accelerates a differentiation pathway, which also affects and slowdown cellular proliferation. Taken together, these data reveal iASPP as a key regulator of epithelial homeostasis.
This manuscript identifies an essential role for the p53 inhibitor iASPP in keratinocyte biology. By regulating two new microRNAs, iASPP controls p63, a key transcriptional regulator for the formation of stratified epithelia.
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